The present invention relates to a nucleic acid sequence encoding a lipolytic enzyme from Fusarium solani, as well as a recombinant method of producing the lipolytic enzyme.
Lipolytic enzymes (such as lipases and phospholipases) are known to be useful, e.g., in baking and detergents.
U.S. Pat. No. 5,990,069 discloses a lipase from a strain of Fusarium solani var. minus. A lipase/phospholipase from Fusarium oxysporum and its sequence are disclosed in WO 98/26057.
The inventors have isolated a gene encoding a lipolytic enzyme from Fusarium solani MUCL 38667 and cloned it into an E. coli strain. Accordingly, the invention provides a DNA sequence encoding a lipolytic enzyme.
The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes a lipolytic enzyme and comprises:
a) the DNA sequence encoding a mature lipolytic enzyme cloned into a plasmid present in Escherichia coli DSM 14361,
b) the DNA sequence encoding a mature lipolytic enzyme shown in SEQ ID NO: 1, or
c) an analogue of the DNA sequence defined in a) or b) which
i) has at least 80% identity with said DNA sequence, or
ii) hybridizes at high stringency with said DNA sequence, its complementary strand or a subsequence thereof.
Other aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector. The invention also provides a recombinant methods of producing the lipolytic enzyme.
A comparison with full-length prior-art sequences shows that the mature amino acid sequence of the lipolytic enzyme from Fusarium solani has 66% identity with the lipase/phospholipase from Fusarium oxysporum described above, and the corresponding DNA sequences show 68% identity.
Genomic DNA Source
The DNA sequence of the invention may be isolated from Fusarium solani MUCL 38667 or from Escherichia coli DSM 14361
MUCL 38667 is available on commercial terms from Mycothxc3xa8que de l""Universitxc3xa9 Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium by referring to U.S. Pat. No. 5,990,069.
E. coli DSM 14361 contains a plasmid with a gene encoding the lipolytic enzyme. It was deposited by the inventors on DSM 14361 under the terms of the Budapest Treaty with the DSMZxe2x80x94Deutshe Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany.
Lipolytic Enzyme
The lipolytic enzyme encoded by the DNA sequence of the invention is able to hydrolyze carboxylic ester bonds and is classified as EC 3.1.1 according to Enzyme Nomenclature 1992, Academic Press, Inc. The enzyme has lipase (triacylglycerol lipase) activity (EC 3.1.1.3) and may also have phospholipase activity.
Further properties of the lipolytic enzyme are described in U.S. Pat. No. 5,990,069.
Recombinant Expression Vector
The expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes. The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.
Production by Cultivation of Transformant
The lipolytic enzyme of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipolytic enzyme, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
The host organism is preferably a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, e.g. a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A. niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S. cerevisiae. The production of the lipolytic enzyme in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).
Hybridization
The hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to a DNA sequence of the invention. The hybridization conditions are described in detail below.
Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5xc3x97SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5xc3x97SSC (Sambrook et al. 1989), 5xc3x97Denhardt""s solution (Sambrook et al. 1989), 0.5% SDS and 100 xcexcg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity greater than 1xc3x97109 cpm/xcexcg) probe for 12 hours at approx. 45xc2x0 C. The filter is then washed two times for 30 minutes in 2xc3x97SSC, 0.5% SDS at a temperature of at least 55xc2x0 C., more preferably at least 60xc2x0 C., more preferably at least 65xc2x0 C., even more preferably at least 70xc2x0 C., especially at least 75xc2x0 C.
Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.
Alignment and Identity
The lipolytic enzyme and the nucleotide sequence of the invention may have identity to the disclosed sequences of at least 85%, particularly at least 90% or at least 95%, e.g. at least 98%.
For purposes of the present invention, alignments of sequences and calculation of identity scores were done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is xe2x88x9212 for proteins and xe2x88x9216 for DNA, while the penalty for additional residues in a gap is xe2x88x922 for proteins and xe2x88x924 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), xe2x80x9cImproved Tools for Biological Sequence Analysisxe2x80x9d, PNAS 85:2444-2448, and W. R. Pearson (1990) xe2x80x9cRapid and Sensitive Sequence Comparison with FASTP and FASTAxe2x80x9d, Methods in Enzymology, 183:63-98).
Lipase Activity (LU)
A substrate for lipase is prepared an emulsion of 5% by volume of tributyrin (glycerin tributyrate) using 0.1% gum Arabic as emulsifier. The hydrolysis of tributyrin at 30xc2x0 C. at pH 7 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 xcexcmol butyric acid/min at the standard conditions. 1 KLU=1000 LU.
Use of Lipolytic Enzyme
The lipolytic enzyme of the invention can be used in various industrial application of lipolytic enzymes, e.g. in baking, detergents, diglyceride synthesis (EP 307154), acidolysis, interesterification (WO 8802775), ester hydrolysis, oil degumming (JP-A 2-153997, U.S. Pat. No. 5,264,367), production of lysolecithin (JP patent 2794574, JP-B 6-087751) and in the process described in PCT/DK 00/00109.
Use in Baking
The lipolytic enzyme of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake. Thus, the lipolytic enzyme can be used in a process for making bread, comprising adding the lipolytic enzyme to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with WO 9404035 and EP 585988.
Use in Detergent
The variant may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg per liter of wash liquor.
The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations. In a laundry detergent, the variant may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup. A laundry detergent composition may be formulated as described in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939 and WO 97/43375.
The detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations. e.g. as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter and Gamble). In a dishwashing composition, the variant may be effective for removal of greasy/oily stains, for prevention of the staining/discoloration of the dishware and plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware.